Microbiota-mediated inflammation and antimicrobial defense in the intestine.

The diverse microbial populations constituting the intestinal microbiota promote immune development and differentiation, but because of their complex metabolic requirements and the consequent difficulty culturing them, they remained, until recently, largely uncharacterized and mysterious. In the last decade, deep nucleic acid sequencing platforms, new computational and bioinformatics tools, and full-genome characterization of several hundred commensal bacterial species facilitated studies of the microbiota and revealed that differences in microbiota composition can be associated with inflammatory, metabolic, and infectious diseases, that each human is colonized by a distinct bacterial flora, and that the microbiota can be manipulated to reduce and even cure some diseases. Different bacterial species induce distinct immune cell populations that can play pro- and anti-inflammatory roles, and thus the composition of the microbiota determines, in part, the level of resistance to infection and susceptibility to inflammatory diseases. This review summarizes recent work characterizing commensal microbes that contribute to the antimicrobial defense/inflammation axis.

IFN-λ suppresses intestinal inflammation by non-translational regulation of neutrophil function.

Interferon-λ (IFN-λ) is a central regulator of mucosal immunity; however, its signaling specificity relative to that of type I interferons is poorly defined. IFN-λ can induce antiviral interferon-stimulated genes (ISGs) in epithelia, while the effect of IFN-λ in non-epithelial cells remains unclear. Here we report that neutrophils responded to IFN-λ. We found that in addition to inducing ISG transcription, IFN-λ (but not IFN-ß) specifically activated a translation-independent signaling pathway that diminished the production of reactive oxygen species and degranulation in neutrophils. In mice, IFN-λ was elicited by enteric viruses and acted on neutrophils to decrease oxidative stress and intestinal damage. Thus, IFN-λ acted as a unique immunomodulatory agent by modifying transcriptional and non-translational neutrophil responses, which might permit a controlled development of the inflammatory process.

The fire within: microbes inflame tumors.

The immune system and the microbiota mutually interact to maintain homeostasis in the intestine. However, components of the microbiota can alter this balance and promote chronic inflammation, promoting intestinal tumor development. We review recent advances in understanding the complex interactions between the microbiota and the innate and adaptive immune systems and discuss their potential to lead us in new directions for understanding cancer biology and treatment.

Epithelial glycosylation in gut homeostasis and inflammation.

Intestinal epithelial cells apically express glycans, especially α1,2-fucosyl linkages, which work as a biological interface for the host-microbe interaction. Emerging studies have shown that epithelial α1,2-fucosylation is regulated by microbes and by group 3 innate lymphoid cells (ILC3s). Dysregulation of the gene (FUT2) encoding fucosyltransferase 2, an enzyme governing epithelial α1,2-fucosylation, is associated with various human disorders, including infection and chronic inflammatory diseases. This suggests a critical role for an interaction between microbes, epithelial cells and ILC3s mediated via glycan residues. In this Review, using α1,2-fucose and Fut2 gene expression as an example, we describe how epithelial glycosylation is controlled by immune cells and luminal microbes. We also address the pathophysiological contribution of epithelial α1,2-fucosylation to pathogenic and commensal microbes as well as the potential of α1,2-fucose and its regulatory pathway as previously unexploited targets in the development of new therapeutic approaches for human diseases.

Gut Mycobiota in Immunity and Inflammatory Disease.

The mammalian intestine is colonized by a wealth of microorganisms-including bacteria, viruses, protozoa, and fungi-that are all integrated into a functional trans-kingdom community. Characterization of the composition of the fungal community-the mycobiota-has advanced further than the much-needed mechanistic studies. Recent findings have revealed roles for the gut mycobiota in the regulation of host immunity and in the development and progression of human diseases of inflammatory origin. We review these findings here while placing them in the context of the current understanding of the pathways and cellular networks that induce local and systemic immune responses to fungi in the gastrointestinal tract. We discuss gaps in knowledge and argue for the importance of considering bacteria-fungal interactions as we aim to define the roles of mycobiota in immune homeostasis and immune-associated pathologies.

Severe Immune-Related Enteritis after In Utero Exposure to Pembrolizumab.

Immune checkpoint blockade has become standard treatment for many types of cancer. Such therapy is indicated most often in patients with advanced or metastatic disease but has been increasingly used as adjuvant therapy in those with early-stage disease. Adverse events include immune-related organ inflammation resembling autoimmune diseases. We describe a case of severe immune-related gastroenterocolitis in a 4-month-old infant who presented with intractable diarrhea and failure to thrive after in utero exposure to pembrolizumab. Known causes of the symptoms were ruled out, and the diagnosis of pembrolizumab-induced immune-related gastroenterocolitis was supported by the results of histopathological assays, immunophenotyping, and analysis of the level of antibodies against programmed cell death protein 1 (PD-1). The infant's condition was successfully treated with prednisolone and infliximab.

Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection.

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3' position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts.

Differentiation and Protective Capacity of Virus-Specific CD8+ T Cells Suggest Murine Norovirus Persistence in an Immune-Privileged Enteric Niche.

Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV-specific tissue-resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV-specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre-existing MNV-specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune-privileged enteric niche.

Attenuation hotspots in neurotropic human astroviruses.

During the last decade, the detection of neurotropic astroviruses has increased dramatically. The MLB genogroup of astroviruses represents a genetically distinct group of zoonotic astroviruses associated with gastroenteritis and severe neurological complications in young children, the immunocompromised, and the elderly. Using different virus evolution approaches, we identified dispensable regions in the 3' end of the capsid-coding region responsible for attenuation of MLB astroviruses in susceptible cell lines. To create recombinant viruses with identified deletions, MLB reverse genetics (RG) and replicon systems were developed. Recombinant truncated MLB viruses resulted in imbalanced RNA synthesis and strong attenuation in iPSC-derived neuronal cultures confirming the location of neurotropism determinants. This approach can be used for the development of vaccine candidates using attenuated astroviruses that infect humans, livestock animals, and poultry.

Rotavirus-mediated DGAT1 degradation: A pathophysiological mechanism of viral-induced malabsorptive diarrhea.

Gastroenteritis is among the leading causes of mortality globally in infants and young children, with rotavirus (RV) causing ~258 million episodes of diarrhea and ~128,000 deaths annually in infants and children. RV-induced mechanisms that result in diarrhea are not completely understood, but malabsorption is a contributing factor. RV alters cellular lipid metabolism by inducing lipid droplet (LD) formation as a platform for replication factories named viroplasms. A link between LD formation and gastroenteritis has not been identified. We found that diacylglycerol O-acyltransferase 1 (DGAT1), the terminal step in triacylglycerol synthesis required for LD biogenesis, is degraded in RV-infected cells by a proteasome-mediated mechanism. RV-infected DGAT1-silenced cells show earlier and increased numbers of LD-associated viroplasms per cell that translate into a fourfold-to-fivefold increase in viral yield (P < 0.05). Interestingly, DGAT1 deficiency in children is associated with diarrhea due to altered trafficking of key ion transporters to the apical brush border of enterocytes. Confocal microscopy and immunoblot analyses of RV-infected cells and DGAT1-/- human intestinal enteroids (HIEs) show a decrease in expression of nutrient transporters, ion transporters, tight junctional proteins, and cytoskeletal proteins. Increased phospho-eIF2α (eukaryotic initiation factor 2 alpha) in DGAT1-/- HIEs, and RV-infected cells, indicates a mechanism for malabsorptive diarrhea, namely inhibition of translation of cellular proteins critical for nutrient digestion and intestinal absorption. Our study elucidates a pathophysiological mechanism of RV-induced DGAT1 deficiency by protein degradation that mediates malabsorptive diarrhea, as well as a role for lipid metabolism, in the pathogenesis of gastroenteritis.

Viral gastroenteritis.

Since the discovery of norovirus in 1972 as a cause of what was contemporarily known as acute infectious non-bacterial gastroenteritis, scientific understanding of the viral gastroenteritides has continued to evolve. It is now recognised that a small number of viruses are the predominant cause of acute gastroenteritis worldwide, in both high-income and low-income settings. Although treatment is still largely restricted to the replacement of fluid and electrolytes, improved diagnostics have allowed attribution of illness, enabling both targeted treatment of individual patients and prioritisation of interventions for populations worldwide. Questions remain regarding specific genetic and immunological factors underlying host susceptibility, and the optimal clinical management of patients who are susceptible to severe or prolonged manifestations of disease. Meanwhile, the worldwide implementation of rotavirus vaccines has led to substantial reductions in morbidity and mortality, and spurred interest in vaccine development to diminish the impact of the most prevalent viruses that are implicated in this syndrome.

Disease-specific plasma protein profiles in patients with fever after traveling to tropical areas.

Fever is common among individuals seeking healthcare after traveling to tropical regions. Despite the association with potentially severe disease, the etiology is often not determined. Plasma protein patterns can be informative to understand the host response to infection and can potentially indicate the pathogen causing the disease. In this study, we measured 49 proteins in the plasma of 124 patients with fever after travel to tropical or subtropical regions. The patients had confirmed diagnoses of either malaria, dengue fever, influenza, bacterial respiratory tract infection, or bacterial gastroenteritis, representing the most common etiologies. We used multivariate and machine learning methods to identify combinations of proteins that contributed to distinguishing infected patients from healthy controls, and each other. Malaria displayed the most unique protein signature, indicating a strong immunoregulatory response with high levels of IL10, sTNFRI and II, and sCD25 but low levels of sCD40L. In contrast, bacterial gastroenteritis had high levels of sCD40L, APRIL, and IFN-γ, while dengue was the only infection with elevated IFN-α2. These results suggest that characterization of the inflammatory profile of individuals with fever can help to identify disease-specific host responses, which in turn can be used to guide future research on diagnostic strategies and therapeutic interventions.

Human norovirus cultivation systems and their use in antiviral research.

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases, affecting all age groups. Despite its clinical needs, no approved antiviral therapies are available. Since the discovery of HuNoV in 1972, studies on anti-norovirals, mechanism of HuNoV infection, viral inactivation, etc., have been hampered by the lack of a robust laboratory-based cultivation system for HuNoV. A recent breakthrough in the development of HuNoV cultivation systems has opened opportunities for researchers to investigate HuNoV biology in the context of de novo HuNoV infections. A tissue stem cell-derived human intestinal organoid/enteroid (HIO) culture system is one of those that supports HuNoV replication reproducibly and, to our knowledge, is most widely distributed to laboratories worldwide to study HuNoV and develop therapeutic strategies. This review summarizes recently developed HuNoV cultivation systems, including HIO, and their use in antiviral studies.

A mathematical, classical stratification modeling approach to disentangling the impact of weather on infectious diseases: A case study using spatio-temporally disaggregated Campylobacter surveillance data for England and Wales.

Disentangling the impact of the weather on transmission of infectious diseases is crucial for health protection, preparedness and prevention. Because weather factors are co-incidental and partly correlated, we have used geography to separate out the impact of individual weather parameters on other seasonal variables using campylobacteriosis as a case study. Campylobacter infections are found worldwide and are the most common bacterial food-borne disease in developed countries, where they exhibit consistent but country specific seasonality. We developed a novel conditional incidence method, based on classical stratification, exploiting the long term, high-resolution, linkage of approximately one-million campylobacteriosis cases over 20 years in England and Wales with local meteorological datasets from diagnostic laboratory locations. The predicted incidence of campylobacteriosis increased by 1 case per million people for every 5° (Celsius) increase in temperature within the range of 8°-15°. Limited association was observed outside that range. There were strong associations with day-length. Cases tended to increase with relative humidity in the region of 75-80%, while the associations with rainfall and wind-speed were weaker. The approach is able to examine multiple factors and model how complex trends arise, e.g. the consistent steep increase in campylobacteriosis in England and Wales in May-June and its spatial variability. This transparent and straightforward approach leads to accurate predictions without relying on regression models and/or postulating specific parameterisations. A key output of the analysis is a thoroughly phenomenological description of the incidence of the disease conditional on specific local weather factors. The study can be crucially important to infer the elusive mechanism of transmission of campylobacteriosis; for instance, by simulating the conditional incidence for a postulated mechanism and compare it with the phenomenological patterns as benchmark. The findings challenge the assumption, commonly made in statistical models, that the transformed mean rate of infection for diseases like campylobacteriosis is a mere additive and combination of the environmental variables.

Homeostasis and inflammation in the intestine.

The gut is home to our largest collection of microbes. The ability of the immune system to coevolve with the microbiota during postnatal life allows the host and microbiota to coexist in a mutually beneficial relationship. Failure to achieve or maintain equilibrium between a host and its microbiota has negative consequences for both intestinal and systemic health. In this Review, we consider the many cellular and molecular methods by which inflammatory responses are regulated to maintain intestinal homeostasis and the disease states that can ensue when this balance is lost.

Nanozyme-Enhanced Probiotic Spores Regulate the Intestinal Microenvironment for Targeted Acute Gastroenteritis Therapy.

Antibiotic therapeutics to combat intestinal pathogen infections often exacerbate microbiota dysbiosis and impair mucosal barrier functions. Probiotics are promising strategies, because they inhibit pathogen colonization and improve intestinal microbiota imbalance. Nevertheless, their limited targeting ability and susceptibility to oxidative stress have hindered their therapeutic potential. To tackle these challenges, Ces3 is synthesized by in situ growth of CeO2 nanozymes with positive charges on probiotic spores, facilitating electrostatic interactions with negatively charged pathogens and possessing a high reactive oxygen species (ROS) scavenging activity. Importantly, Ces3 can resist the harsh environment of the gastrointestinal tract. In mice with S. Typhimurium-infected acute gastroenteritis, Ces3 shows potent anti-S. Typhimurium activity, thereby alleviating the dissemination of S. Typhimurium into other organs. Additionally, owing to its O2 deprivation capacity, Ces3 promotes the proliferation of anaerobic probiotics, reshaping a healthy intestinal microbiota. This work demonstrates the promise of combining antibacterial, anti-inflammatory, and O2 content regulation properties for acute gastroenteritis therapy.

Detection of Rotavirus in Respiratory Specimens From Bangladeshi Children Aged <2 Years Hospitalized for Acute Gastroenteritis.

To examine the potential for respiratory transmission of rotavirus, we systematically assessed if rotavirus RNA is detectable by real-time quantitative reverse transcription-polymerase chain reaction from nasal and oropharyngeal swab specimens of Bangladeshi children with acute rotavirus gastroenteritis. Forehead swabs were collected to assess skin contamination. Among 399 children aged <2 years hospitalized for gastroenteritis during peak rotavirus season, rotavirus RNA was detected in stool, oral, nasal and forehead swab specimens of 354 (89%). A subset was genotyped; genotype was concordant within a child's specimen set and several different genotypes were detected across children. These findings support possible respiratory transmission of rotavirus and warrant further investigation.

Clinical Impact of Multiplex Molecular Diagnostic Testing in Children With Acute Gastroenteritis Presenting to an Emergency Department: A Multicenter Prospective Study.

BACKGROUND: Multiplex molecular diagnostic panels have greatly enhanced detection of gastrointestinal pathogens. However, data on the impact of these tests on clinical and patient-centered outcomes are limited. METHODS: We conducted a prospective, multicenter, stepped-wedge trial to determine the impact of multiplex molecular testing at 5 academic children's hospitals on children presenting to the emergency department with acute gastroenteritis. Caregivers were interviewed on enrollment and 7-10 days after enrollment to determine symptoms, risk factors, subsequent medical visits, and impact on family members. During the pre-intervention period, diagnostic testing was performed at the clinician's discretion . During the intervention period, multiplex molecular testing was performed on all children, with results available to clinicians. The primary outcome was return visits to a healthcare provider within 10 days of enrollment. RESULTS: Potential pathogens were identified by clinician-ordered tests in 19 of 571 (3.3%) in the pre-intervention period compared with 434 of 586 (74%) in the intervention period; clinically relevant pathogens were detected in 2.1% and 15%, respectively. In the multivariate model, the intervention was associated with a 21% reduction in the odds of any return visit (odds ratio, 0.79; 95% confidence interval, .70-.90) after adjusting for potential confounders. Appropriate treatment was prescribed in 11.3% compared with 19.6% during the intervention period (P = .22). CONCLUSIONS: Routine molecular multiplex testing for all children who presented to the ED with acute gastroenteritis detected more clinically relevant pathogens and led to a 21% decrease in return visits. Additional research is needed to define patients most likely to benefit from testing. Clinical Trials Registration. NCT02248285.

Emergence of Novel Norovirus GII.4 Variant.

We detected a novel GII.4 variant with an amino acid insertion at the start of epitope A in viral protein 1 of noroviruses from the United States, Gabon, South Africa, and the United Kingdom collected during 2017-2022. Early identification of GII.4 variants is crucial for assessing pandemic potential and informing vaccine development.

Emerging Variants of Canine Enteric Coronavirus Associated with Outbreaks of Gastroenteric Disease.

A 2022 canine gastroenteritis outbreak in the United Kingdom was associated with circulation of a new canine enteric coronavirus closely related to a 2020 variant with an additional spike gene recombination. The variants are unrelated to canine enteric coronavirus-like viruses associated with human disease but represent a model for coronavirus population adaptation.